A variety of gene delivery technologies can be used to express antigens within somatic tissues, resulting in systemic humoral and cellular immune responses. This observation has led to the development of polynucleotide vaccine preparations for stimulation of systemic immunity.
In the late 1980s, it became apparent that the direct injection of plasmid DNA or mRNA into a variety of tissues would result in polynucleotide uptake and expression of encoded proteins (U.S. Pat. No. 5,580,859). These data resulted in the development of a gene transfer-based vaccine paradigm. Realizing the importance of cytotoxic cellular immune responses, it was further proposed that genetic immunization could be used to stimulate both humoral and cellular immunity (U.S. Pat. No. 5,589,446). Subsequent experimentation demonstrated that direct gene transfer into tissues can elicit complete systemic immune responses to a variety of antigens (Ulmer et al., Science 259:1745-1749, 1993; Wang et al, Proc Natl Acad Sci USA 90(9):4156-4160, 1993). A number of studies have since extended the DNA vaccine model to include a wide variety of gene delivery systems and therapeutic applications.
More recently, research concerning potential therapeutic uses for naked gene expression vectors has focused on enhancing gene expression through use of different promotors, delivery vehicles and routes of administration (see, e.g., Stribling, et al., Proc. Natl. Acad. Sci. USA, 89:11277-11281, 1992 [expression following aerosol delivery of a gene occurred with use of a liposomal delivery system]; and, Tang, et al., Nature, 356:152-154, 1992 [injection with a vaccine "gun" of an hGH plasmid coupled to colloidal gold beads]).
The immune system can be divided into two functionally independent compartments: the systemic compartment, which is represented by the bone marrow, spleen, and lymph nodes, and the mucosal compartment, which is represented by lymphoid tissues in mucosae and external secretory glands. A consequence of this compartmentalization is that systemic routes of immunization are usually of limited value for the prevention of mucosa-contracted diseases. Studies performed in animal models and with humans have convincingly demonstrated that the level of protection against diseases of the respiratory, genital, or intestinal tract correlate better with the levels of antibodies in corresponding external secretions than in serum.
In general, current polynucleotide vaccine strategies focus on the stimulation of systemic humoral and/or cellular immune responses to specific antigens. Unfortunately, the majority of infectious disease is acquired via mucosal surfaces, and systemic polynucleotide vaccine strategies do not typically elicit mucosal immune responses. For instance, a common route for the initial acquisition of HIV involves passage of the virus across a mucosal surface. Disruption of the mucosa allows the virus to reach the underlying lymphoid cells in the lamina propria. Secretory immunoglobulins A (sIgA) may function as a first line of defense against such infections, preventing attachment and transmission through the mucosa. In addition, it is believed that sIgA can inhibit viral replication within infected epithelial cells.
Like the systemic immune compartment, the common mucosal immune system requires mechanisms for selective switching between the expansion of effector cells and the induction of tolerance. Inappropriate induction of mucosal immune responses can result in clinical syndromes including food and respiratory allergies (Holt and McMenamin, Clin Exp Allergy 19(3): 255-62, 1989; Brandtzaeg et al.(1993) The serologic and mucosal immunologic basis of celiac disease. Immunophysiology of the Gut. Bristol-Meyers squibb/Mead Johnson Nutrition Symposia Eds. W. A. Walker, P. R. Harmatz and B. K. Wershil. London., Academic press. 295-333). The mechanism(s) involved in switching between induction or suppression of mucosal immune responses remain to be resolved, but may involve antigen sampling and presentation by either specialized inductor tissues (stimulation) or MHC Class II.sup.+ mucosal epithelial cells (tolerance in gut (Brandtzaeg et al., Gastroenterology 97(6): 1562-84, 1989), hypersensitivity or tolerance in lung (Kalb et al., Am J Respir Cell Mol Biol 4(4): 320-9. 1991)). These studies illustrate the complex nature of mucosal immune response regulation, and support the hypothesis that selection of different tissues for transfection or transduction with a mucosal polynucleotide vaccine may result in profound differences in the resulting pattern of immune response.
Mucosal immunity typically involves both cellular cytotoxic as well as antibody-mediated responses. Production of secreted IgA is a widely accepted surrogate marker for complete mucosal immune responses. Efforts to raise sIgA using current polynucleotide vaccination methods have been inconclusive. The pathways by which mucosal immune response can be elicited have not been fully characterized. Mucosal antigen presentation can be associated with either immunologic stimulation or induction of tolerance. Hence, there are multiple hypotheses for the absence of IgA in lung lavage and nasal secretion samples collected after mucosal expression of an antigenic protein via DNA vaccination. Alternative hypotheses include inadequate gene transfer and expression and inappropriately targeted gene transfer and expression.
Discrimination between these hypotheses requires an efficient gene delivery and expression vector system which is replication-defective. Such system is required for testing the importance of anatomic targeting for enabling mucosal immune responses. Therefore, one system meeting these criteria involves an engineered Semliki Forest Virus (SFV) alphaviruses (Liljestrom and Garoff, Biotechnology (N Y) 9(12): 1356-61. 1991). Alphaviruses are positive strand RNA viruses, and hence the RNA genomes of these agents produce infectious particles upon transfection (Zhou et al., Vaccine 12(16): 1510-4. 1994) The Semliki Forest virus (SFV) has been engineered to yield a vector system based on a genomic SFV cDNA inserted into an SP6 RNA promoter plasmid. The resulting plasmid has been modified by deletion of the SFV structural genes to allow insertion of a heterologous cDNA as part of the SFV replicon. After incorporation of the cDNA of interest, in vitro SP6 transcription of plasmid DNA results in mRNA preparations which encode both the recombinant protein as well as the SFV replicase, which can be assembled into viral particles and used to infect cells. Typically after infection, the polymerase and recombinant protein become a major fraction of total cellular protein (Liljestrom and Garoff 1991). Subsequent cytotoxicity typically limits expression of the recombinant protein to four to seven days.
One potential complication of all virally-derived gene transfer systems is the development of replication-competent helper virus. In alphavirus-derived systems, this occurs via polymerase strand crossover between the mRNA which encodes the protein of interest, and a trans helper mRNA which provides packaging proteins used to produce defective particles for transduction. By modifying the viral spike protein encoded by SFV, conditionally infectious particles which require activation by chymotrypsin can be produced, and this modification has reduced the production of replication-competent helper particles to undetectable levels (Berglund et al., Biotechnology (N Y) 11(8): 916-20. 1993). Therefore, gene delivery and expression remain confined to the site of initial innoculation. Recombinant SFV mRNA and defective particles have been shown to stimulate a strong systemic immune response to a recombinant protein after genetic vaccination (Zhou et al. 1994; Zhou, Berglund et al. 1995)
Thus, the need exists for a safe and effective means of introducing nucleotides that will express in vivo a peptide or protein which can induce mucosal immunity to vaccinate a host against, for example, respiratory illnesses, sexually transmitted diseases, and other diseases which are initially contracted through the mucosa.
In the instant invention, we have exploited this efficient transduction and expression activity of recombinant SFV particles to test the hypothesis that mucosal genetic vaccines will require gene transfer methods which target specific mucosal-associated inductor tissues such as the oropharyngeal Waldeyer's ring or intestinal Peyer's patches.